The long term goal of this project is to understand the molecular basis of mutation in a higher plant by analyzing the mutant alleles of the maize waxy(Wx) gene. Several transposable element mutations in maize are characterized by a leaky phenotype; analysis of the leaky wx-m9 Ds allele revealed that the Ds element was spliced from Wx pre-mRNA. Subsequent studies revealed that three different classes of Ds elements, associated with three different wx mutations, are spliced from pre-mRNA in a similar manner. Whereas Group I and II introns are thought to be evolving into mobile DNA, the Ds elements may be mobile DNA evolving into introns. To determine the cis-requirements for Ds splicing we will assess the ability of in vitro mutagenized Dsl elements to be processed following electroporation into maize protoplasts. We will also determine whether the multiple 5' splice sites found in the terminus of all Ds element have been conserved because they are required for splicing or for transposition. Finally, we will use EMS mutagenesis in an attempt to isolate second-site mutations that simultaneously suppress the mutant phenoytpes of leaky Ds alleles at two loci. Similar mutations in Drosophila and yeast have led to the identification of proteins involved in transcription initiation and RNA processing. In the second part of the proposal we will focus on 4 novel retrotransposons responsible for 6 stable wx mutations. We know very little about plant retrotransposons; our Wx elements almost double the number of plant elements identified to date. We propose to analyze these elements by determining their sequence, species distribution and identifying element specific transcripts. We have determined that four of the retro-induced mutations are leaky due to the processing of element sequences from either Wx introns or exons. Two of these four mutations are expressed in the endosperm but not in the pollen. We hypothesize that tissue specific alternative splicing is responsible for the observed phenotypes and propose to test this notion by using S 1 analysis and RNAase protection to assess the splicing patterns in each tissue. In a similar mutant hunt as described above for the Ds mutations, we will use EMS mutagenesis in an attempt to isolate second-site suppressors of the retro-induced wx mutations. We have characterized all of the large lesions among the wx mutations. In order to derive a complete and unbiased picture of the molecular basis of mutation in maize, we will define the remaining 11 mutations that have been localized on the genetic map but have no obvious physical lesion. To this end we will exploit the correlation between our physical map and the genetic map to localize the lesions to restricted regions of the Wx gene and isolate and sequence these lesions following polymerase chain reaction (PCR) amplification.